The long-term goal of this research project is to define the sepcificity, mechanism and in vivo role of the ubiquitin dependent protein degradation system. This soluble multienzyne system has been partially characterized in lysates of rabbit reticuloytes. Ubiquitin, a small, universally distributed and highly conserved protein is covalently attached to proteins during their degradation by the system. These conjugates are proposed to be steady state intermediates with specific proteases binding to the ubiquitin portion and degrading the attached protein. Thus, ubiquitin is a cofactor and not itself degraded. ATP is required to make the conjugates and this system may partially explain the universal energy dependence of in vivo protein degradation. We will chemically modify the ubiquitin to examine the cofactor specificity of the overall reaction and the individual partial reactions. Of special interest will be derivatives which are functional in some but not all of the partial reactions. We will also chemically modify substrate proteins to begin to assess those features which are recognized by the specific enzymes of this system. Liver extracts will be examined for the presence of substrates, inhibitors, or individual enzymes of the system. Finally, affinity adsorbants of immobilized ubiquitin will be sunthesized in order to purify the ubiquitin binding proteins and enzymes. This broad-based approach will begin to characterize the substrate and cofactor specificity of the system. In addition the development of improved methods of assay and isolation of the enzymes should clarify the individual reactions. The mechanisms and control of intracellular protein degradation remain unclear, although they are of obvious importance. Changes in the amount of proteins through control of their degradation rate may be important for metabolic control, response to horomonal stimuli, development, differentiation and aging. The observations that individual proteins have discreet half lives demands specificity in proteolysis. The fact that ubiquitin and energy dependent proteolytic systems are universally distributed suggests that the ubiquitin-dependent system may be extremely important in intracellular protein degradation in all cells.